Simultaneous detection of target protein and target nucleic acids in a single cell

ABSTRACT

Methods, reagents, and kits for detection and analysis of nucleic acids are provided. The methods can be used in conjunction with a proximity extension assay for protein detection to provide a multiplex assay to detect both nucleic acids (e.g., RNA) and proteins.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/775,960, filed Internationally on Mar. 14, 2014, which is a USNational Phase of PCT Application No. PCT/US2014/028751, filed Mar. 14,2014, which claims benefit of U.S. provisional Application No.61/799,559, filed Mar. 15, 2013, herein incorporated by reference forall purposes.

FIELD OF THE INVENTION

The invention relates to an amplification-based detection system that issufficiently sensitive for detection of nucleic acids, e.g., RNA in asingle cell. The method can be used in conjunction with a proximityextension assay for protein detection to provide a multiplex assay todetect both nucleic acids and proteins.

BACKGROUND

Detection and quantification of protein and nucleic acids fromindividual cells is desirable, but difficult to achieve because of theminute amount of material present in a single cell. Further, unlike bulksamples, a single cell cannot be divided into portions to separatelyanalyze protein and nucleic acid levels. Although single moleculedetection techniques or mass spectrometry may provide methods forachieving single cell analysis, such methods are expensive. Recently, anassay, the Proximity Extension Assay (PEA) has been developed that issensitive enough to detect picogram quantities of protein (see, e.g.,Lundberg et al., Nucl. Acids Res. 2011 August; 39(15):e102; epub 2011Jun. 6, incorporated by reference herein). In one approach, the PEAemploys a pair of antibodies, each having a oligonucleotide attached toit. The oligonucleotides contain regions that complement one another.When the antibodies bind to a target protein, the oligonucleotides arein close enough proximity so that complementary regions from eacholigonucleotide hybridize to one another. The addition of a DNApolymerase results in extension of the hybridized oligonucleotides. Theextension products can then be detected or quantified.

BRIEF DESCRIPTION OF THE INVENTION

In various aspects, the invention includes, but is not limited to, thefollowing embodiments.

In one aspect the invention provides a method of detecting a targetnucleic acid, typically RNA, in a sample, the method comprising (a)incubating in a reaction mixture: i) a sample comprising a targetnucleic acid; and ii) a pair of proximity probes comprising a first andsecond probe, where: the first probe comprises a target binding (TB)segment that hybridizes to a first target (T) segment of the targetnucleic acid, and an interacting (I) segment at the 3′ end of the probe,wherein the I segment is complementary to an I segment at the 3′ end ofthe second probe; and the second probe comprises a TB segment thathybridizes to a second, non-overlapping T segment of the target nucleicacid that is in close proximity to the first T segment, and an I segmentat the 3′ end, wherein the 3′ sequence is complementary to the I segmentof the first probe, wherein

the reaction mixture is incubated under conditions in which the TBsegment of the first probe hybridizes to the first T segment of thetarget nucleic acid and the TB segment of the second probe hybridizes tothe second T segment of the target nucleic acid, thereby allowing the Isegment of the first probe to hybridize to the I segment of the secondprobe to form a duplex comprising the I segments of the first and secondprobe;(b) adding a DNA polymerase and maintaining the reaction mixture underconditions in which the first and/or second probe is extended to obtaina first extended product;(c) amplifying the extended product, or a subregion thereof, in anamplification reaction mixture comprising a pair of amplificationprimers that amplify the first extended product, or subregion thereof;(d) detecting the amplicon obtained in (c).

In some embodiments, the step of detecting the amplicon comprisesquantifying the amplicon, e.g., using a qPCR reaction.

In some embodiments, the sample is a single cell.

In some embodiments, one of the members of the proximity pair is blockedat the 3′ end so that only one probe is extended in step (b).

In some embodiments, the DNA polymerase employed in the extension stephas 3′ exonuclease activity. In some embodiments, the polymerase usedfor the amplification step is different from the polymerase used for theextension step. For example, the polymerase used for the amplificationstep may be thermostable whereas the polymerase used for the extensionstep may not be thermostable.

In some embodiments, the method further comprises detecting a targetprotein in the sample. In such embodiments, the method furthercomprises:

incubating the sample in the reaction mixture of (a) with a pair ofprotein-detecting proximity probes comprising a first and a secondprotein-detecting proximity probe where:

the first protein-detecting probe comprises a first antibody that bindsto the target protein joined to a first polynucleotide that comprises anI segment at the 3′ end that is complementary to an I segment on the 3′end of the second probe; and

the second protein-detecting probe comprises a second antibody thatbinds to the target protein joined to a second polynucleotide thatcomprises an I segment complementary to the I segment at the 3′ end ofthe first polynucleotide;

wherein binding of the first antibody to the target protein and bindingof the second antibody to the target protein allows the I segment of thefirst protein proximity probe to hybridize to the I segment of thesecond protein proximity probe to form a duplex which is extended instep (b) to provide a second extended product;amplifying the second extended product, or subregion thereof, in theamplification reaction of (c) using a set of primers that amplify thesecond extended product or subregion thereof; anddetecting the amount of amplicon from amplification of the secondextended product or subregion thereof.

In some embodiments, the step of detecting the amount comprisesquantifying the amount of amplicon, e.g., using a qPCR reaction.

in some embodiments, the reaction is a multiplex reaction in whichmultiple RNAs are detected.

In a further aspect, the invention includes a use of a proximity probepair for the detection/quantification of a target nucleic acid in asample; or a use of proximity probe pairs for detection/quantificationof target nucleic acids and proteins in a sample. In some embodiments,the invention provides a use of a proximity probe pair for thedetection/quantification of a target nucleic acid in a sample; or a useof proximity probe pairs for detection/quantification of target nucleicacids and proteins in a sample, wherein one of the members of theproximity probe pair is blocked.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates an embodiment of a nucleic acid proximity extensionassay to detect a target RNA. In this embodiment, the 3′ end of eachmember of the probe pair is extendible.

FIG. 2 illustrates another embodiment of a nucleic acid proximityextension assay to detect a target RNA. In this embodiment, the 3′ endof one of the probes is blocked.

DETAILED DESCRIPTION 1. Definitions and Terminology

As used herein, a “sequence” means a nucleic acid base sequence of apolynucleotide. Unless otherwise indicated or apparent from context,bases or sequence elements are presented in the order 5′ to 3′ as theyappear in a polynucleotide.

A “polynucleotide” or “nucleic acid” includes any form of RNA or DNA,including, for example, genomic DNA; complementary DNA (cDNA), which isa DNA representation of messenger RNA (mRNA), usually obtained byreverse transcription of mRNA; and DNA molecules produced syntheticallyor by amplification. Polynucleotides may include chimeric molecules andnucleic acids comprising non-standard bases (e.g., inosine).Polynucleotides may be single-stranded or double-stranded.

The term “oligonucleotide” is used herein to refer to a nucleic acidthat is relatively short, generally shorter than 200 nucleotides, moreparticularly, shorter than 100 nucleotides or shorter than 50nucleotides. Typically, oligonucleotides are single-stranded DNAmolecules.

A “target polynucleotide” or “target nucleic acid” is a polynucleotidethat comprises a target sequence. In a double-stranded targetpolynucleotide the target sequence is on one strand and the complementof the target sequence is on the other strand. A “target RNA” is an RNAthat comprises a target sequence.

The term “segment,” refers to a sequence or subsequence in apolynucleotide, such as a segment having a particular function, e.g.,probe-binding segment, primer-binding segment, indexing sequence, alsoreferred to herein as a “tag sequence”, and others listed herein.Individual segments may have any length consistent with their intendedfunction, such as, without limitation, lengths in the range of 10-100nucleotides, 10-70 nucleotides, 14-50 nucleotides, and 14-35nucleotides.

A “target sequence” is a nucleic acid sequence detected in an assay. Inmost cases a target sequence of interest is predefined (i.e., sequenceis known prior to analysis). In other cases the complete target sequenceis not known, but is defined as the sequence that is amplified byprimers of known sequence. A target sequence may be found in DNA(including genomic, mitochondrial, viral, synthetic and cDNA), in RNA,or in amplifiable synthetic analogs thereof.

As used herein, the term “complementary” refers to the capacity forprecise pairing between two nucleotides. I.e., if a nucleotide at agiven position of a nucleic acid is capable of hydrogen bonding with anucleotide of another nucleic acid, then the two nucleic acids areconsidered to be complementary to one another at that position. A“complement” may be an exactly or partially complementary sequence.Complementarity between two single-stranded nucleic acid molecules maybe “partial,” in which only some of the nucleotides bind, or it may becomplete when total complementarity exists between the single-strandedmolecules. Two oligonucleotides are considered to have “complementary”sequences when there is sufficient complementarity that the sequenceshybridize (forming a double stranded region) under assay conditions. Thedegree of complementarity between nucleic acid strands has significanteffects on the efficiency and strength of hybridization between nucleicacid strands. Two sequences that are partially complementary may have,for example, at least 90% identity, or at least 95%, 96%, 97%, 98%, or99% identity sequence over a sequence of at least 7 nucleotides, moretypically over a sequence of 10-30 nucleotides, often over a sequence of14-25 nucleotides, and sometimes over a longer sequence (e.g., 26-100nucleotides in length). It will be understood that the 3′ base of aprimer sequence will desirably be perfectly complementary tocorresponding bases of the target nucleic acid sequence to allow primingto occur. A first sequence or segment is “substantially complementary”to a second sequence of segment when a polynucleotide consisting of thefirst sequence is sufficiently complementary to specifically hybridizeto a polynucleotide consisting of the second sequence. For illustration,hybridization conditions are salt concentrations less than about 1.0 Msodium ion, typically about 0.01 to 1.0 M sodium ion at pH 7.0 to 8.3,and temperatures at least about 30° C. for polynucleotides 10 to 50nucleotides in length and at least about 60° C. for longer probes (e.g.,greater than 50 nucleotides). Typically, specific hybridization willoccur when there is at least about 55% base complementary over a stretchof at least 14-25 nucleotides, preferably at least 65%, more preferablyat least 75%, more preferably at least 85%, more preferably at least90%, and most preferably at least 95%. The prime symbol [′] is used toindicate a perfectly or substantially complementary sequence.

The terms “anneal”, “hybridize” or “bind,” in reference to twopolynucleotide sequences, segments or strands, are used interchangeablyand have the usual meaning in the art. Two complementary sequences(e.g., DNA and/or RNA) anneal or hybridize by forming hydrogen bondswith complementary bases to produce a double-stranded polynucleotide ora double-stranded region of a polynucleotide.

Two sequences or segments in a polynucleotide are “adjacent” or“contiguous” if there is no intervening sequence or non-nucleotidelinker separating them. In some contexts, “non-adjacent” refers to twoprobe-binding sequences separated from each other by an interveningtarget sequence.

A “primer” is an oligonucleotide or polynucleotide comprising a sequencethat is complementary to, and capable of hybridizing to, a targetsequence, or the complement thereof. In general, “primer” means an“extendible primer” that can prime template-dependent DNA synthesis. Insome cases a primer is extended by a DNA-dependent DNA polymerase.

A proximity probe can include a “nucleotide tag”. The term “nucleotidetag” is used herein to refer to a predetermined nucleotide sequence thatis incorporated into a proximity probe to facilitate identifying thetarget molecule in a multiplex action.

A proximity probe typically comprises DNA, but may also includepolyribonucleotides (containing D-ribose), and any other type of nucleicacid that is an N- or C-glycoside of a purine or pyrimidine base, aswell as other polymers containing normucleotidic backbones, for example,polyamide (e.g., peptide nucleic acids (PNAs)) and polymorpholino(commercially available from the Anti-Virals, Inc., Corvallis, Oreg., asNeugene) polymers, and other synthetic sequence-specific nucleic acidpolymers providing that the polymers contain nucleobases in aconfiguration which allows for base pairing and base stacking, such asis found in DNA and RNA.

The terms “multiplex” and “multiplexing” refer to assays in which two ormore primer sets are used to amplify two or more distinct targetsequences in the same amplification reaction mixture.

As used herein, “amplification” of a nucleic acid sequence has its usualmeaning, and refers to in vitro techniques for enzymatically increasingthe number of copies of a target sequence. Amplification methods includeboth asymmetric methods (in which the predominant product issingle-stranded) and conventional methods (in which the predominantproduct is double-stranded).

The terms “amplicon” and “amplification product” are usedinterchangeably and have their usual meaning in the art. Thegrammatically singular term, “amplicon,” can refer to many identicalcopies of an amplification product. Moreover, reference to an “amplicon”encompasses both a molecule produced in an amplification step andidentical molecules produced in subsequent amplification steps (such as,but not limited to, amplification products produced in subsequent roundsof a PCR amplification). Moreover, the term “amplification may refer tocycles of denaturation, annealing and extension, and does not requiregeometric or exponential increase of a sequence.

A “amplification reaction mixture” is the solution in which anamplification reaction takes place and may comprise one or more oftarget polynucleotides, primers, polymerase, ligase, amplificationreagents, amplicons, buffering agents, nuclease inhibitors, divalentcations, dNTPs, and/or other components known in the art foramplification.

The term “qPCR” is used herein to refer to quantitative real-timepolymerase chain reaction (PCR), which is also known as “real-time PCR”or “kinetic polymerase chain reaction.”

As used herein, a “sample” refers to a composition containing a targetpolynucleotide. A “sample” may also contain a target protein. Exemplarysamples include cells and cell lysates (e.g., eukaryotic cells, humancells, animal cells, plant cells, stem cells, blood cells, lymphocytes,bacterial cells, recombinant cells and cells infected with a pathogen.tissue samples), viruses, environmental samples (e.g., water samples),food samples, forensic samples, plant samples, blood samples and thelike. “Cell lysates” includes partially purified cell fractions.

A “reagent” refers broadly to any agent used in a reaction, other thanthe analyte (e.g., nucleic acid being analyzed). Illustrative reagentsfor a nucleic acid amplification reaction include, but are not limitedto, buffer, metal ions, polymerase, reverse transcriptase, primers,template nucleic acid, nucleotides, labels, dyes, nucleases, and thelike. Reagents for enzyme reactions include, for example, substrates,cofactors, buffer, metal ions, inhibitors, and activators.

The term “label,” as used herein, refers to any atom or molecule thatcan be used to provide a detectable and/or quantifiable signal. Inparticular, the label can be attached, directly or indirectly, to anucleic acid or protein. Suitable labels that can be attached to probesinclude, but are not limited to, radioisotopes, fluorophores,chromophores, mass labels, electron dense particles, magnetic particles,spin labels, molecules that emit chemiluminescence, electrochemicallyactive molecules, enzymes, cofactors, and enzyme substrates.

2. Overview

In one aspect, the invention provides proximity extension methods fordetecting a target nucleic acid in a sample. The method is typicallyused concurrently with a proximity extension assay for detecting levelsof protein in the same sample. Typically, the target nucleic acid thatis detected is an RNA. In some embodiments, the target may besingle-stranded DNA, such as a single-stranded DNA virus.

In some embodiments, a target nucleic acid (e.g., RNA) is detected in asample by a process in which a pair of proximity probes are hybridizedto the target nucleic acid (e.g., RNA). Each member of a pair ofproximity probes (“PPP”) comprises a target binding segment (“TB”segment), often at or near the 5′ end of the probe, that hybridizes to apredefined segment of the target nucleic acid (“T” segment). Each memberof a PPP comprises an interaction segment (“I segment”), often at ornear the 3′ end of the probe, where the interaction segment of onemember of the PPP is complementary to the interaction segment present inthe other member of the PPP. The sequences of the proximity probes areselected or designed so that TB segments in each member of the proximityprobe pair bind to different regions of the target nucleic acid (i.e.,different, nonoverlapping, T segments), where the T segments are locatedin sufficiently close proximity, and the PPP's have sufficient length,so that the I segments can interact when the proximity probes arehybridized to the target RNA.

Binding of the probes to the target nucleic acid allows the I segmentsof the PPP to hybridize. A DNA polymerase is then added that extends thehybridized probes at their 3′ ends, extending the portion of the PPPdimer that is double stranded. Hybridization of the I segments andextension of the duplex results in an extended product that can then bedetected in an amplification reaction. Typically, each member of theproximity probe pair also comprises an amplification primer binding site(APBS) segment. Generally, each of the two probes of a PPP have adifferent APBS. Thus, when the proximity probes are hybridized via theirI segments and extended by the polymerase, a double-stranded (orpartially double-stranded) polynucleotide is generated, having a pair ofAPBSs. It will be recognized that each proximity probe has a singleAPBS, and the extension step results in a polynucleotide with bothAPBSs.

The methods of the invention can be conveniently used in a multiplexassay format. For example, if two or more target molecules, e.g., two ormore target nucleic acids such as two different RNA targets, are to bedetected, the products can be detected in a single reaction usingmultiple pairs of proximity probes, each of which forms an extensionproduct that is unique. Similarly, when a nucleic acid, e.g., RNA, and aprotein are to be detected in the same reaction, proximity probe pairsfor the nucleic acid and protein, each of which forms a unique extensionproduct, are used concurrently in a singled reaction. An assay of theinvention can thus be readily multiplexed to evaluate the presence oramounts of multiple target molecules in a sample.

Amplification primers are used to amplify the extended product. Thedetermination of the presence, absence, quantity, or relative amount ofthe amplified product is indicative of the presence, absence, quantity,or relative amount of the target sequence in the initial sample.

In some embodiments, the amount of extended product is quantified in aqPCR reaction. A variety of other amplification systems may be used, asdiscussed below.

The methods for detecting a target nucleic acid, e.g., RNA, may also beused in conjunction with a proximity extension assay that detectsprotein present in a sample, as further detailed below.

3. Nucleic Acid Proximity Probe Pairs

This invention employs a proximity probe pair to detect a target nucleicacid, typically a target RNA, such as an mRNA. In other embodiments, aproximity probe pair can be used to detect a target DNA, such as viralDNA that may be present in a sample. Each member of the proximity probepair comprises the following regions: a TB segment, an I segment, and anAPBS segment. The TB segment is complementary to and binds a T segmentof the target nucleic acid sequence. One member of the proximity probepair binds to T segment on the target nucleic acid and the other memberbinds to a different, non-overlapping T segment on the target nucleicacid. The sites are in close proximity, i.e., such that hybridization ofthe TB regions to the target nucleic acid allows the complementary Iregions to hybridize. Typically the probe binding sites are separated byfewer than 100 bases, often fewer than 50 bases, e.g., 40, 30, 20, or 15nucleotides or less. In some embodiments two sequences or segments in apolynucleotide are considered to be “in close proximity” when they areseparated by from 10 to 50 bases.

The TB segment of a proximity probe is located at or near the 5′ end ofthe probe. For example, the TB segment may be positioned within 2-10nucleotides of the nucleotide at the 5′ end of the proximity probe. Thesize of the TB segment typically ranges anywhere from 10 to 100nucleotides in length. In some embodiments, the TB segment is less than50 nucleotides in length, and may be less than 20 or 10 nucleotides inlength. For example, a TB segment may be from 5 to 20 or 10 to 40nucleotides in length. These ranges are illustrative guidelines but arenot intended to limit the invention.

The I region of a proximity probe is located at or near the 3′ end ofthe probe such that the I region is available to hybridize to thecomplementary I region of the other member of the probe pair when theproximity probe pairs are hybridized to the target nucleic acid. Intypical embodiments, an I segment is designed such that uponhybridization with the I segment of the other member of the proximitypair, there are no 3′ non-base-paired nucleotides. However, otherembodiments are also contemplated. For example, the 3′ end, i.e., thathas the free 3′ hydroxyl group, of one of the proximity probes may notbe included in the I segment that binds to the complementary I segmentof the other member of the proximity probe pair, thus leavingnon-base-paired nucleotides at the 3′ end. Use of a polymerase having a3′ exonuclease activity will permit the extension of the probe that hasthe 3′ non-based-paired nucleotides. In other embodiments, only one ofthe probes may be extended. Thus, a probe may be designed to havenon-base-paired nucleotides at the 3′ end. In some embodiments, one ofthe probes may be modified at the 3′ end to prevent extension.

Typically, the I segment is less than 20 nucleotides in length. Forexample, the I segment may be from 6 to 12 nucleotides in length, e.g.,6, 7, 8, 9, 10, 11 or 12 nucleotides in length.

In typical embodiments, each member of the proximity probe pair alsocomprises an amplification primer binding site. Upon extension of theproximity probes, polynucleotide molecules in which both APBS sequencesare present are produced. Extended polynucleotides having bothamplification primer binding sites permits the amplification of theextension product using amplification primers that bind to the primerbinding site. Alternatively, one member of the proximity probe pair mayhave an amplification primer binding site. A second amplification primerbinding site, may for example, be created upon hybridization of the Iregions of the proximity probes. In other embodiments only one member ofthe proximity probe pair may be extended. The extended probe cancomprise two amplification primer binding sites to permit amplificationof the extended product.

Probes are typically designed to avoid areas of secondary structure inthe RNA. For example, one of skill can use known computer programs toprovide a model of a structure of a target RNA. The TB region of theprobe may then be designed to avoid regions of secondary structure suchas hairpins, stems, and the like. Probes that are complementary to atarget RNA segment can be designed using software readily available inthe art, e.g., Primer 3 (Whitehead Institute for Biomedical Research).

In some embodiments, one of the members of the probe pair may be blockedto prevent extension. For example, one of the probes may have a modifiedbase at the 3′ end that prevents extension of the probe. In someembodiments, the 3′ nucleotide may be phosphorylated. In otherembodiments, the 3′ end may have a modified nucleotide such as athiophosphate-modified nucleotide, a 2′-OMe-CE phosphoramidite-modifiednucleotide, or another extension-blocking nucleotide known in the art.In some embodiments, one or more nucleotides that prevent extension of aprobe may be located upstream of the 3′ end such that extension does notoccur beyond that point. For example, a linker may be used to blockextension of the probe.

The concentration of probes added to the reaction mixture to hybridizeto target RNA is typically in the range of 1 nM to 100 nM. The time andtemperature of incubation of the proximity probes with the sample canvary, depending on the particular probes used. The time should besufficient so that the probes bind to the target nucleic acid and thatthe 3′ ends hybridize. In illustrative embodiments, the time ofincubation may be from anywhere from about 10 minutes to about 1-2hours, or longer, e.g., 12 to 24 hours. The temperature at which thereaction is conducted can vary, depending on the probes employed anddepending on whether other molecules, such as protein, are also detectedin a multiplex assay. When only RNA is detected, typical incubationstemperatures of the probe with the target can range from about 55° C. toabout 70° C. Often, the incubation temperature is from about 60° C. toabout 70° C., e.g., the incubation temperature is 61° C., 62° C., 63°C., 64° C., 65° C., 66° C., 67° C., 68° C., 69° C., or 70° C. When theassay is a multiplex assay that also detects protein, incubationstemperatures are typically lower, for example from about 35° C. to about45° C. In some embodiments, the temperature is in a range from about 37°C. to about 42° C. Illustrative reaction conditions for a multiplexreaction comprising detecting both target RNA and a target proteinscomprise a probe concentration of 1 nM and incubation at 37° C. for onehour. Illustrative reaction conditions for detecting target RNA onlycomprise a probe concentration of 1 nM and incubation at 65° C. for 18to 24 hours.

In some embodiments, the reaction additionally comprises components thatcan stabilize probe:RNA hybridization. An example of such a component isRNase H in which the RNase activity has been inactivated, or asingle-stranded nucleic acid binding protein us as T4 gp32.

4. Extension Reaction

The extension reaction is typically carried out after the hybridizationof the probes to the target molecules. Reagents such as nucleotides anda DNA polymerase are included in the extension reaction. Any DNApolymerase can be used. In some embodiments the DNA polymerase has 3′exonuclease activity. Examples of such polymerases include T4 DNApolymerase, T7 DNA polymerase, Phi29 (ϕ29) DNA polymerase, DNApolymerase I, Klenow fragment of DNA polymerase I, Pyrococcus furiosus(Pfu) DNA polymerase, and Pyrococcus woesei (Pwo) DNA polymerase.

In other embodiments, a DNA polymerase that lacks 3′ to 5′ exonucleaseactivity may be employed. Such polymerases include polymerases such asTaq or ΔTaq polymerase.

The temperature at which the extension reaction is conducted depends onthe nature of the polymerase employed. For example, reactions employingthermostable polymerases may be conducted at temperatures above 40degrees Celsius. A temperature is employed, however, that allows thehybridized proximity probes to remain hybridized to the target nucleicacid and for the 3′ end of the probes to stably hybridize.

The polymerase may be added to the assay along with the proximity probesor may be added following addition of the proximity probes. In someembodiments, the polymerase is added after a period of incubation of theproximity probes with the target polynucleotide.

5. Protein-Detecting Proximity Probe Pairs

In some embodiments, the detection of a target nucleic acid of interest,e.g., an RNA of interest, is performed simultaneously with detection ofa protein of interest in the same reaction mixture. Accordingly, in someembodiments, the method comprises performing a proximity extension assayas described herein for detecting one or more specific RNAs andperforming a proximity extension assay to detect one or more specificproteins. Proximity extension assays for detecting proteins are wellknown in the art (see, for example, Lundberg et al. Nucl. Acids Res. 39:e102, 2011; and WO2012/104261, each of which is incorporated byreference) and any such assay can be used.

In an illustrative assay that can be used in a multiplex assay with anucleic acid proximity extension assay, a protein proximity probe pair,i.e., a proximity probe pair for detecting a protein of interest,comprises one probe that includes a nucleic acid binding segment (i.e,an I segment) linked to an antibody that binds to the protein ofinterest. The second probe in the pair includes a nucleic acid bindingsegment, (i.e., an I segment) that is complementary to the I segment ofthe first probe and is also linked to an antibody that binds to thetarget protein of interest. Upon binding of the antibodies to the targetprotein, the I segments hybridize.

The antibodies used for the protein proximity probes may be polyclonalor monoclonal antibodies, or fragments of antibodies. Further, theantibodies linked to each member of the protein proximity probe pair mayhave the same binding specificity or differ in their bindingspecificities. The present invention further contemplates use ofvariations of this assay, e.g., that are described in WO2012/104261. Forexample, the probes may each be linked to their respective antibody atthe 5′ end, or one probe may be linked at the 5′ end and the other atthe 3′ end.

As noted above, upon binding of the antibodies of the protein proximityprobe pair to the target protein, the 3′ ends hybridize to form adouble-stranded nucleic acid that has at least one 3′ OH that can beextended by a polymerase as described above. As in the case of thenucleic acid proximity probe pair (for detecting the nucleic acid ofinterest, e.g., an RNA of interest), the proximity probes for theprotein hybridize such that a unique sequence is created to serve as asequence tag, which can be used as an identifier.

The extension reaction is performed at a temperature appropriate for theselected polymerase and under conditions in which the antibodies remainbound to the target proteins such that the 3′ complementary ends of theprobe pairs can hybridize. In an assay in which both nucleic acidproximity probe pairs and protein proximity probe pairs are used todetect a target nucleic acid and a target protein in the same reaction,the extension reaction is performed at a temperature appropriate for theselected polymerase and under conditions in which the T segments of thenucleic acid proximity probes remain bound to the target nucleic acidand the antibodies for the protein proximity probes remain bound to thetarget proteins so that for each of the proximity probe pairs, the 3′complementary segments can hybridize.

6. Amplification and Detection of Amplified Products

The extended products obtained from the extension reactions aresubjected to an amplification reaction to obtain an amplified productthat can be detected and quantified, as desired. Design parameters ofvarious amplification reactions are well known. Examples of referencesproviding guidance are provided below. In some embodiments theamplification reaction uses the same polymerase that is used in theextension assay, optionally without addition of more polymerase. In someembodiments the amplification reaction uses a polymerase that isdifferent from the polymerase used for the extension assay. For example,in some embodiments, a polymerase having a 3′ exonuclease activity maybe used in the extension reactions and a Taq polymerase may be used inthe amplification reaction.

In some embodiments, an amplification reaction may employ a hot-startpolymerase. For example, a recombinant Taq DNA polymerase complexed withan antibody that inhibits polymerase activity at ambient temperaturesmay be used. The polymerase is active after a PCR denaturation step.

Any method of detection and/or quantitation of nucleic acids can be usedin the invention to detect and/or quantify amplification products. Inparticular embodiments, real-time quantification methods are used. Forexample, “quantitative real-time PCR” methods can be used to determinethe quantity of an amplified product present in a sample by measuringthe amount of amplification product formed during the amplificationprocess itself. This method of monitoring the formation of amplificationproduct involves the measurement of PCR product accumulation at multipletime points. The amount of amplified product reflects the amount oftarget nucleic acid or target protein present in the sample.

Fluorogenic nuclease assays are one specific example of a real-timequantitation method that can be used successfully in the methodsdescribed herein. This method of monitoring the formation ofamplification product involves the continuous measurement of PCR productaccumulation using a dual-labeled fluorogenic oligonucleotide probe—anapproach frequently referred to in the literature as the “TaqMan®method.” See U.S. Pat. No. 5,723,591; Heid et al, 1996, Real-timequantitative PCR Genome Res. 6:986-94, each incorporated herein byreference in their entireties for their descriptions of fluorogenicnuclease assays. It will be appreciated that while “TaqMan® probes” arethe most widely used for qPCR, the invention is not limited to use ofthese probes; any suitable probe can be used.

Other detection/quantitation methods that can be employed in the presentinvention include FRET and template extension reactions, molecularbeacon detection, Scorpion detection, and Invader detection.

FRET and template extension reactions utilize a primer labeled with onemember of a donor/acceptor pair and a nucleotide labeled with the othermember of the donor/acceptor pair. Prior to incorporation of the labelednucleotide into the primer during a template-dependent extensionreaction, the donor and acceptor are spaced far enough apart that energytransfer cannot occur. However, if the labeled nucleotide isincorporated into the primer and the spacing is sufficiently close, thenenergy transfer occurs and can be detected. These methods are describedin U.S. Pat. No. 5,945,283 and PCT Publication WO 97/22719.

With molecular beacons, a change in conformation of the probe as ithybridizes to a complementary region of the amplified product results inthe formation of a detectable signal. The probe itself includes twosections: one section at the 5′ end and the other section at the 3′ end.These sections flank the section of the probe that anneals to the probebinding site and are complementary to one another. One end section istypically attached to a reporter dye and the other end section isusually attached to a quencher dye. In solution, the two end sectionscan hybridize with each other to form a hairpin loop. In thisconformation, the reporter and quencher dye are in sufficiently closeproximity that fluorescence from the reporter dye is effectivelyquenched by the quencher dye. Hybridized probe, in contrast, results ina linearized conformation in which the extent of quenching is decreased.Thus, by monitoring emission changes for the two dyes, it is possible toindirectly monitor the formation of amplification product. Probes ofthis type and methods of their use are described further, for example,by Piatek et al. (1998) Nat. Biotechnol. 16: 359-363; Tyagi, and Kramer(1996) Nat. Biotechnol, 14: 303-308; and Tyagi, et α/.(1998) Nat.Biotechnol. 16:49-53. [0124] The Scorpion detection method is described,for example, by Thelwell et al. (2000) Nucleic Acids Res., 28: 3752-3761and Solinas et al. (2001) Nucleic Acids Res., 29(20): e96. Scorpionprimers are fluorogenic PCR primers with a probe element attached at the5′-end via a PCR stopper. They are used in real-time amplicon-specificdetection of PCR products in homogeneous solution. Two different formatsare possible, the “stem-loop” format and the “duplex” format. In bothcases the probing mechanism is intramolecular. The basic elements ofScorpions in all formats are: (i) a PCR primer; (ii) a PCR stopper toprevent PCR read-through of the probe element; (iii) a specific probesequence; and (iv) a fluorescence detection system containing at leastone fluorophore and quencher. After PCR extension of the Scorpionprimer, the resultant amplicon contains a sequence that is complementaryto the probe, which is rendered single-stranded during the denaturationstage of each PCR cycle. On cooling, the probe is free to bind to thiscomplementary sequence, producing an increase in fluorescence, as thequencher is no longer in the vicinity of the fluorophore. The PCRstopper prevents undesirable read-through of the probe by Taq DNApolymerase. [0125] Invader assays (Third Wave Technologies, Madison,Wis.) are used particularly for SNP genotyping and utilize anoligonucleotide, designated the signal probe, that is complementary tothe target nucleic acid (DNA or RNA) or polymorphism site. A secondoligonucleotide, designated the Invader Oligo, contains the same 5′nucleotide sequence, but the 3′ nucleotide sequence contains anucleotide polymorphism. The Invader Oligo interferes with the bindingof the signal probe to the target nucleic acid such that the 5′ end ofthe signal probe forms a “flap” at the nucleotide containing thepolymorphism. This complex is recognized by a structure specificendonuclease, called the Cleavase enzyme. Cleavase cleaves the 5′ flapof the nucleotides. The released flap binds with a third probe bearingFRET labels, thereby forming another duplex structure recognized by theCleavase enzyme. This time, the Cleavase enzyme cleaves a fluorophoreaway from a quencher and produces a fluorescent signal.

As noted above, various amplification and reaction methods may be usedto detect the extended product. Thus, amplification according to thepresent invention encompasses any means by which at least a part of theextended product is copied, typically in a template-dependent manner,including without limitation, a broad range of techniques for amplifyingnucleic acid sequences, either linearly or exponentially. Illustrativemeans for performing an amplifying step include ligase chain reaction(LCR), ligase detection reaction (LDR), ligation followed by Q-replicaseamplification, PCR, primer extension, strand displacement amplification(SDA), hyperbranched strand displacement amplification, multipledisplacement amplification (MDA), nucleic acid strand-basedamplification (NASBA), two-step multiplexed amplifications, rollingcircle amplification (RCA), and the like, including multiplex versionsand combinations thereof. Descriptions of such techniques can be foundin, among other sources, Ausbel et al.; PCR Primer: A Laboratory Manual,Diffenbach, Ed., Cold Spring Harbor Press (1995); The ElectronicProtocol Book, Chang Bioscience (2002); Msuih et al., J. Clin. Micro.34:501-07 (1996); The Nucleic Acid Protocols Handbook, R. Rapley, ed.,Humana Press, Totowa, N.J. (2002); Abramson et al., Curr OpinBiotechnol. 1993 February; 4(I): 41-7, U.S. Pat. No. 6,027,998; U.S.Pat. No. 6,605,451, Barany et al., PCT Publication No. WO 97/31256; Wenzet al., PCT Publication No. WO 01/92579; Day et al., Genomics, 29(1):152-162 (1995), Ehrlich et al., Science 252:1643-50 (1991); Innis etal., PCR Protocols: A Guide to Methods and Applications, Academic Press(1990); Favis et al., Nature Biotechnology 18:561-64 (2000); and Rabenauet al., Infection 28:97-102 (2000); Belgrader, Barany, and Lubin,Development of a Multiplex Ligation Detection Reaction DNA Typing Assay,Sixth International Symposium on Human Identification, 1995 (availableon the world wide web at:promega.com/geneticidproc/ussymp6proc/blegrad.html-); LCR KitInstruction Manual, Cat. #200520, Rev. #050002, Stratagene, 2002;Barany, Proc. Natl. Acad. Sci. USA 88:188-93 (1991); Bi and Sambrook,Nucl. Acids Res. 25:2924-2951 (1997); Zirvi et al., Nucl. Acid Res. 27:e40i-viii (1999); Dean et al., Proc Natl Acad Sci USA 99:5261-66 (2002);Barany and Gelfand, Gene 109: 1-11 (1991); Walker et al., Nucl. AcidRes. 20: 1691-96 (1992); Polstra et al., BMC Inf. Dis. 2:18-(2002); Lageet al., Genome Res. 2003 February; 13(2): 294-307, and Landegren et al.,Science 241: 1077-80 (1988), Demidov, V., Expert Rev Mol Diagn. 2002November; 2(6): 542-8., Cook et al., J Microbiol Methods. 2003 May;53(2): 165-74, Schweitzer et al., Curr Opin Biotechnol. 2001 February;12(I): 21-7, U.S. Pat. Nos. 5,830,711, 6,027,889, 5,686,243, PCTPublication No. WO0056927A3, and PCT Publication No. WO9803673A1.

As used herein, the term “amplification” includes isothermalamplification methods. Isothermal amplification uses a constanttemperature rather than cycling through denaturation andannealing/extension steps. Some means of strand separation, e.g., anezyme, is used in place of thermal denaturation. Examples of isothermalamplification include: hyperbranched strand displacement amplification(Groathouse, N., et al. (2006) “Isothermal Amplification and MolecularTyping of the Obligate Intracellular Pathogen Mycobacterium lepraeIsolated from Tissues of Unknown Origins” J. Clin. Micro. 44 (4):1502-1508); helicase-dependent amplification (Vincent, M., et al. (2004)“Helicase-dependent isothermal DNA amplification” EMBO Rep. 5 (8):795-800); multiple displacement amplification (MDA; Luthra, R., andMedeiros, J. (2004) “Isothermal Multiple Displacement Amplification” JMol Diagn. 6 (3): 236-242); loop-mediated isothermal amplification(Notomi, T., et al. (2000) Nucleic Acids Research 28 (1); PAN-AC (David,F. and Turlotte, E., (1998) “An Isothermal Amplification Method” C.R.Acad. Sci Paris, Life Science 321 (1): 909-14); strand displacementamplification (SDA; Nycz, C, et al. (1998) Analytical Biochemistry 259(2): 226-234); rolling circle amplification (RCA; Lizardi, P., et al.,(1998)“Mutation detection and single-molecule counting using isothermalrolling-circle amplification” Nature Genetics 19: 225-232); nucleic acidstrand-based amplification (NASBA; Van Der Vliet, G., et al. (1993)“Nucleic acid sequence-based amplification (NASBA) for theidentification of mycobacteria” Journal of General Microbiology 139(10): 2423-2429; and recombinase polymerase amplification (U.S. Pat.Nos. 7,485,428; 7,399,590; 7,270,981; and 7,270,951, each of which isincorporated by reference in its entirety and specifically for itsdescription of recombinase polymerase amplification).

In embodiments in which fluorophores are used as labels, many suitablefluorophores are known. Examples of fluorophores that can be usedinclude, but are not limited to, rhodamine, cyanine 3 (Cy 3), cyanine 5(Cy 5), fluorescein, Vic™, Liz™, Tamra™, 5-Fam™, 6-Fam™, and Texas Red(Molecular Probes). (Vic™, Liz™, Tamra™, 5-Fam™, 6-Fam™ are allavailable from Applied Biosystems, Foster City, Calif.).

In embodiments in which quenchers are also used for detection ofamplified products, useful quenchers include, but are not limited totetramethylrhodamine (TAMRA), DABCYL (DABSYL, DABMI or methyl red)anthroquinone, nitrothiazole, nitroimidazole, malachite green, BlackHole Quenchers®, e.g., BHQ1 (Biosearch Technologies), Iowa Black® or ZENquenchers (from Integrated DNA Technologies, Inc.), TIDE Quencher 2(TQ2) and TIDE Quencher 3 (TQ3) (from AAT Bioquest).

PCR and fluorescence detection can conveniently be performed using asystem such as the BioMark™ System (Fluidigm Corporation, South SanFrancisco).

7. Samples

Any target nucleic acid can be detected using the proximity extensionprobe assays of the invention. In typical embodiments, the targetnucleic acid is an RNA molecule. The targets can include, for example,nucleic acids associated with pathogens, such as viruses, bacteria,protozoa, or fungi; RNAs, e.g., those for which over- orunder-expression is indicative of disease, those that are expressed in atissue- or developmental-specific manner; or those that are induced byparticular stimuli. In some embodiments, the methods comprisesconcurrent detection of both RNA and protein targets is a sample using anucleic acid proximity extension assay as described herein and a proteinproximity extension assay

Samples comprising a nucleic acid, e.g., RNA, or nucleic acid andprotein of interest can be obtained from biological sources and preparedusing conventional methods known in the art. In particular, samples tobe analyzed in accordance with the methods described herein obtainedfrom any source, including bacteria, protozoa, fungi, viruses,organelles, as well higher organisms such as plants or animals,particularly mammals, and more particularly humans. Other samples can beobtained from environmental sources (e.g., pond water, air sample), fromman-made products (e.g., food), from forensic samples, and the like.Samples can be obtained from cells, bodily fluids (e.g., blood, a bloodfraction, urine, etc.), or tissue samples by any of a variety ofstandard techniques. Illustrative samples include samples of plasma,serum, spinal fluid, lymph fluid, peritoneal fluid, pleural fluid, oralfluid, and external sections of the skin; samples from the respiratory,intestinal genital, and urinary tracts; samples of tears, saliva, bloodcells, stem cells, or tumors. For example, samples can be obtained froman embryo or from maternal blood. Samples can also be obtained from liveor dead organisms or from in vitro cultures. Illustrative samples caninclude single cells, paraffin-embedded tissue samples, and needlebiopsies.

The assays of the invention can be carried out on single cells or apopulation of cells (i.e., two or more cells). In some embodiments anassay is conducted using nucleic acids and proteins obtained from asingle cell, or small number (fewer than 10, or fewer than 5) of cells.In one approach employing a single cell, the cell is isolated and lysed;and reagents, e.g., proximity extension probes, extension reagents,polymerases, amplification reagents are added directly to the lysate toperform the detection assay. In some embodiments, the assay, theisolation of macromolecules from single cells, or both are carried outusing a microfluidic device. Microfluidic systems for isolating andobtaining macromolecules from single cells and/or conducting assaysusing the macromolecules are known. An exemplary device is the C1™Single-Cell Auto Prep System which is commercially available fromFluidigm Corp. 7000 Shoreline Court, Suite 100, South San Francisco,Calif.). The C1™ Single-Cell Auto Prep System isolates single cells,lyses them, and carries out a series of reactions from the lysate (e.g.,cDNA synthesis, nucleic acid amplification, etc.). Other devices aredescribed in U.S. patent application Ser. No. 13/781,292 filed Feb. 28,2013, entitled “Methods, Systems, And Devices For Multiple Single-CellCapturing And Processing Using Microfluidics”; and U.S. ProvisionalApplication No. 61/852,135 filed Mar. 15, 2013, entitled “Methods AndDevices For Analysis Of Defined Multicellular Combinations,” both ofwhich are incorporated by reference in their entirety for all purposes.Optionally the C1™ Single-Cell Auto Prep System may be used inconjunction with Fluidigm's BioMark™ HD System (Fluidigm Corp. 7000Shoreline Court, Suite 100, South San Francisco, Calif.). U.S. patentapplication Ser. No. 13/781,292 filed Feb. 28, 2013 is incorporatedherein in its entirety all purposes.

Other devices for manipulation of single cells include the following(none of which are admitted to be prior art): Sims et al., 2007,“Analysis of single mammalian cells on-chip” Lab Chip 7: 423-440;Wheeler et al., 2003, “Microfluidic device for single-cell analysis”Anal Chem 75: 3581-3586; Skelley et al., 2009 “Microfluidic control ofcell pairing and fusion” Nat Methods 6: 147-152; Marcus et al., 2006,“Microfluidic single-cell mRNA isolation and analysis” Anal Chem 78:3084-3089; Bontoux et al., 2008 “Integrating whole transcriptome assayson a lab-on-a-chip for single cell gene profiling” Lab Chip 8: 443-450;Zhong et al., 2008 “A microfluidic processor for gene expressionprofiling of single human embryonic stem cells” Lab Chip 8: 68-74;Wheeler 2003 “Microfluidic Device for Single-Cell Analysis Anal. Chem.”75: 3581-3586; and White et al., Aug. 23, 2011 “High-throughputmicrofluidic single-cell RT-qPCR PNAS” Vol. 108, 34:13999-14004; each ofthe aforelisted publications is incorporated herein by reference.

Additional methods for amplifying and detecting amplified products aredescribed in U.S. Pat. Pub. Nos. 2012-0115143 (“Universal Probe AssayMethods”), US 2012-0288857 (“Multifunctional Probe-Primers”), US2013-0045881 (“Probe Based Nucleic Acid Detection”); and copendingcommonly owned International Patent Application No. PCT/US2012/065376(“NUCLEIC ACID DETECTION USING PROBES”) and International PCTApplication No. PCT/US2007/063229 (“COOPERATIVE PROBES AND METHODS OFUSING THEM”), each of which is expressly incorporated by reference forall purposes.

10. Kits

Kits according to the invention include one or more reagents useful forpracticing one or more assay methods of the invention. A kit generallyincludes a package with one or more containers holding the reagent(s)(e.g., nucleic acid proximity extension probes and optionally, proteinproximity extension probes), as one or more separate compositions. Insome embodiments, the probes may be provided as an admixture where thecompatibility of the reagents will allow. The kit can also include othermaterial(s) that may be desirable from a user standpoint, such as abuffer(s), a diluent(s), a standard(s), and/or any other material usefulin sample processing, washing, or conducting any other step of theassay.

Kits according to the invention generally include instructions forcarrying out one or more of the methods of the invention. Instructionsincluded in kits of the invention can be affixed to packaging materialor can be included as a package insert. While the instructions aretypically written or printed materials they are not limited to such. Anymedium capable of storing such instructions and communicating them to anend user is contemplated by this invention. Such media include, but arenot limited to, electronic storage media (e.g., magnetic discs, tapes,cartridges, chips), optical media (e.g., CD ROM), RF tags, and the like.As used herein, the term “instructions” can include the address of aninternet site that provides the instructions.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims.

In addition, all other publications, patents, and patent applicationscited herein are hereby incorporated by reference in their entirety forall purposes.

The invention claimed is:
 1. A kit for performing a multiplexed assay ofRNA and protein from a sample, the kit comprising: a) at least one pairof protein-detecting proximity probes, wherein each pair ofprotein-detecting proximity probes comprises: a first protein-detectingprobe comprising a first antibody that binds to a target protein joinedto a first polynucleotide that comprises an I segment at the 3′ end thatis complementary to an I segment on the 3′ end of a second probe; and asecond protein-detecting probe comprising a second antibody that bindsto the target protein joined to a second polynucleotide that comprisesan I segment complementary to the I segment at the 3′ end of the firstpolynucleotide of the first probe; wherein binding of the first antibodyto the target protein and binding of the second antibody to the targetprotein allows the I segment of the first protein proximity probe tohybridize to the I segment of the second protein proximity probe to forma duplex; b) a polymerase for extending the duplex to provide anextended product; and c) primers that together amplify the extendedproduct, or subregion thereof; and amplify two or more target nucleicacid sequences in the same reaction mixture.
 2. The kit of claim 1,wherein the primers are packaged as an admixture in the samecomposition.
 3. The kit of claim 1, further comprising one or morelabels for detecting amplicons produced in step c).
 4. The kit of claim1, further comprising reagents to isolate single cells.
 5. The kit ofclaim 1, further comprising reagents to lyse single cells.
 6. The kit ofclaim 1, further comprising a microfluidic device for isolating andlysing single cells.
 7. The kit of claim 1, wherein the kit comprisescompatible probes provided in admixture.
 8. The kit of claim 1, furthercomprising reagents for performing a quantitative amplificationreaction.
 9. The kit of claim 8, wherein the quantitative amplificationreaction is qPCR.
 10. The kit of claim 1, wherein the polymerase is aDNA polymerase having 3′ exonuclease activity.
 11. The kit of claim 1,further comprising a DNA polymerase for performing step (c) that isdifferent from the DNA polymerase employed in the extension reaction ofstep (b).
 12. The kit of claim 11, wherein the DNA polymerase forperforming step (c) is a thermostable polymerase.
 13. The kit of claim1, further comprising reagents for detecting a target RNA sequence andan amplicon obtained by amplifying an extended product of a nucleic acidproximity extension reaction.
 14. The kit of claim 1, wherein RNA of acell in the sample comprises the target nucleic acid sequences.
 15. Amethod of evaluating a single cell for RNA and protein of interest, themethod comprising performing a multiplexed assay on a single cell todetect RNA and protein with a kit comprising: a) at least one pair ofprotein-detecting proximity probes, wherein each pair ofprotein-detecting proximity probes comprises: a first protein-detectingprobe comprising a first antibody that binds to a target protein joinedto a first polynucleotide that comprises an I segment at the 3′ end thatis complementary to an I segment on the 3′ end of a second probe; and asecond protein-detecting probe comprising a second antibody that bindsto the target protein joined to a second polynucleotide that comprisesan I segment complementary to the I segment at the 3′ end of the firstpolynucleotide of the first probe; wherein binding of the first antibodyto the target protein and binding of the second antibody to the targetprotein allows the I segment of the first protein proximity probe tohybridize to the I segment of the second protein proximity probe to forma duplex; b) a polymerase; and c) primers that together amplify theextended product or subregion thereof and amplify two or more targetnucleic acid sequences in the same reaction mixture.